Gene Electrotransfer via Conductivity‐Clamped Electric Field Focusing Pivots Sensori‐Motor DNA Therapeutics: “A Spoonful of Sugar Helps the Medicine Go Down”

Abstract Viral vectors and lipofection‐based gene therapies have dispersion‐dependent transduction/transfection profiles that thwart precise targeting. The study describes the development of focused close‐field gene electrotransfer (GET) technology, refining spatial control of gene expression. Integration of fluidics for precise delivery of “naked” plasmid deoxyribonucleic acid (DNA) in sucrose carrier within the focused electric field enables negative biasing of near‐field conductivity (“conductivity‐clamping”–CC), increasing the efficiency of plasma membrane molecular translocation. This enables titratable gene delivery with unprecedently low charge transfer. The clinic‐ready bionics‐derived CC‐GET device achieved neurotrophin‐encoding miniplasmid DNA delivery to the cochlea to promote auditory nerve regeneration; validated in deafened guinea pig and cat models, leading to improved central auditory tuning with bionics‐based hearing. The performance of CC‐GET is evaluated in the brain, an organ problematic for pulsed electric field‐based plasmid DNA delivery, due to high required currents causing Joule‐heating and damaging electroporation. Here CC‐GET enables safe precision targeting of gene expression. In the guinea pig, reporter expression is enabled in physiologically critical brainstem regions, and in the striatum (globus pallidus region) delivery of a red‐shifted channelrhodopsin and a genetically‐encoded Ca2+ sensor, achieved photoactivated neuromodulation relevant to the treatment of Parkinson's Disease and other focal brain disorders.


Figures
delivery of neurotrophin or control (eGFP) DNA to the cochleae of deafened cats, supporting long-term hearing using bilateral cochlear implants Fig. S7: Sustained regeneration of the peripheral spiral ganglion neurites in the cochleae of deafened cats supported by chronic cochlear implant stimulation following CC-GET delivery of neurotrophin-encoding DNA Fig.S8: Sharpening of inferior colliculus spatial tuning curves with cochlear CC-GET delivery of BDNF -NT3 neurotrophin DNA followed by chronic cochlear implant stimulation in the deafened cat Tables Table S1: Pulse parameters and resistivity for in vivo guinea pig cochlea DNA electrotransfer using the clinical CC-GET device Table S2: Pulse parameters and resistivity for in vivo cat cochlea DNA electrotransfer using the clinical CC-GET device Table S3: Pulse parameters and resistivity for in vivo DNA electrotransfer in guinea pig brain using the clinical CC-GET device

Electric Field Recordings
To establish the effect of conductivity -clamping on the GET electric field, voltage potentials were measured adjacent to the GET array (modified from an 8 electrode research cochlear implant (CI) array, Cochlear Limited, Australia, part number Z60274) (after [1] ).This was done in either non-conductive (10% sucrose), or conductive (0.9% sodium chloride) solutions, with or without 2 mg/ml salmon sperm DNA sheared to an average length of 2.0 kb (Merk).The GET array electrodes were driven in the 'tandem' configuration, with four ganged anodes and four ganged cathodes, as shown in Figure 1A, using either a DS5 isolated stimulator (Digitimer) for fixed recording electrode position experiments (Figure 1A, B), or an A-M Systems 2200 stimulus isolator for field mapping (Figure 1C, D) to drive 100 ms square wave pulses.Pulses were controlled and voltage and current outputs monitored (five channels at 100 kHz sampling rate) using a Digidata 1440 interface (Axon Instruments) with Clampex software (version 10.2.0.12;Axon Instruments).A custom-built electrode monitoring system was used to provide independent measurements of voltage in the field, and both voltage and current applied at the electrodes.The recording electrode was a custom platinum fine tip electrode, referenced to a platinum ground electrode positioned distal to the GET array and the generated electric field.
Measurements were taken at ~ 95 ms during the 100 ms pulses.Electrodes were positioned using a translational stage (SliceScope Pro 6000; Scientifica, East Sussex, UK) and micromanipulator (ROE-200; Sutter Instrument Company, Novato, CA, USA) with 0.5 mm resolution steps.

HEK293 cell monolayer modelling
The HEK293 cell monolayer was used as a high-throughput in vitro model to establish a reporter-based biomarker of the suprathreshold electric field for GET.Adherent HEK293 cells were maintained and prepared for transfection experiments by seeding onto 18 mm round glass coverslips ~ 24 hr prior to transfection, which was undertaken at ~50% cell confluence, following protocols previously published ( [1][2][3] ).Plasmid DNA resuspended at 2 mg/ml was delivered to the HEK293-coated coverslips in 20-30 µl of 10% sucrose, with or without 0.5 mM NaOH, or saline, with or without 50 mM Tris buffer, or 9% sucrose + 0.09% NaCl + 0.5 mM NaOH.Electric pulse trains were delivered via a DS5 (Digitimer) isolated stimulator using a custom-built waveform control and monitoring software.Transfected coverslips were imaged 2-4 days after GET on a LSM710 confocal microscope (Zeiss) using a 488 nm argon ion laser for GFP and a 561 nm DPSS laser for mCherry.For larger fields of transfection, multiple overlapping tiled images were captured and assembled prior to analysis.Boundaries around the region of transfected cells were delineated and the area of transfection or total fluorescence (sum pixel intensity) calculated using ImageJ (US NIH).

Cochlear implant surgery and pFAR4-BDNF-NT3 CC-GET in cats
At ~ 3 months of age, cats underwent CC-GET prior to bilateral implantation of a HL14 CI electrode array (Cochlear Ltd.).The surgery was performed as previously described [4] with the exception that prior to implantation of each cochlea CC-GET was undertaken using a doubleinsertion technique where the clinical CC-GET array was inserted, GET performed and the CC-GET array was removed prior to insertion of the permanent HL14 array for chronic stimulation as a 'bionic ear' hearing prosthesis.CC-GET was undertaken as described for the guinea pig experiments above, but performed bilaterally with randomization of cochlea receiving the pFAR4-CMVp-eGFP DNA and the contralateral cochlea receiving the pFAR4-BDNF-NT3 DNA.
Clinical CIs and speech processors were switched on two weeks after implantation and chronic stimulation in response to environmental sound was presented continuously, except for brief periods when batteries were replaced.Standard clinical frequency allocation tables were used to program speech processors for monopolar stimulation.Biphasic pluses were delivered to each electrode at 500 pulses per second and had a 25 μs phase interval with an 8 μs interphase gap.Impedance of the intracochlear electrodes was measured weekly throughout the chronic stimulation period using monopolar stimulation from each intracochlear electrode to the external return electrode via Custom Sound EP 2.0 software (Cochlear ltd.).

Neural response telemetry (NRT) electrocochleography recording of electrically evoked compound action potentials (eCAPS)
The level of the CI electrodes were recorded for both ears in awake animals every 2-3 weeks throughout the chronic stimulation period using Custom Sound EP 2.0 software (Cochlear ltd.).NRT stimulation pulses were delivered to each of the 14 electrodes in monopolar stimulation using monopolar stimulation (MP+1) to the external reference electrode at 25 µs/phase biphasic pulses delivered at 80 pulses per second.ECAPs were recorded at 10 CL (Current Level) intervals from a maximum of 250 CL to below threshold.

Terminal inferior colliculus recordings
Approximately six months after CC-GET and cochlear implantation acute electrophysiological recordings were taken in the inferior colliculus (IC) immediately prior to euthanasia as previously described [5] over a 24-72 hour period.The multi-electrode array IC recording site was driven by each of the CI arrays inserted into control eGFP DNA and neurotrophin DNA -CC-GET treated cochleae.In anesthetized cats 32-channel, 100 µm spacing, recording electrodes on a silicon substrate (NeuroNexus Technologies, USA) were inserted into the IC bilaterally to a depth of ~ 3.5 mm.Anesthesia was induced by intramuscular injection of ketamine (20 mg/kg) and subcutaneous injection of xylazil (2 mg/kg) and maintained by intravenous infusion of sodium pentobarbital (3-8 mg/kg/hr).Spike activity was acquired from the 64 electrodes using a Cerebus acquisition system (Blackrock Microsystems, USA), with sampling of amplified and filtered data at a rate of 30 kHz.Bipolar stimulation delivered to adjacent intracochlear electrodes of the CI array was 100 μs phase duration and 50 μs interphase gap, bipolar cathodic-first charge balanced at rate of 4 Hz.This was delivered via an inhouse designed multi-channel stimulus generator at CL ranging between 0 and 265 CL (defined by Cochlear Ltd.) where, current in µA = 17.5*(100 ^ (CL/255).Neural activity was detected based on threshold (4x root-mean-square level) crossings and spikes in a 3 -45 ms post stimulus window.At each recording site, the spike counts were recorded across 10 trials for each stimulation configuration and current level to calculate spontaneous activity.IC spatial turning curve (STC) width was determined by calculating the average threshold value across the first five electrodes of the 32-electrode IC array for each of the CI electrodes, for each cochlea.The STC width was then defined as the length of the array where electrodes had a threshold value below the average threshold across the first five electrodes.

Plasmid DNA
Plasmids maps for each of the seven DNA molecules used are shown in Figure S1.For transfection, plasmids were purified from E. coli using Plasmid Maxiprep Kits (Qiagen) according to the instructions.DNA pellets were resuspended in water for quantification on a Nanodrop Nd-1000 spectrophotometer (ThermoFisher).Quantified DNA was dried using a SpeedVac vacuum concentrator (ThermoFisher) at room temperature and resuspended in isotonic saline or sucrose solutions at the desired concentration for transfection.

Guinea pig tissue collection and analysis
Guinea pigs were euthanized using intraperitoneal injection of pentabarbitol (100 mg/kg).Cochlea were isolated and fixed by perfusion of the cochleae scala with 4% paraformaldehyde in phosphate buffer and submerged in 4% paraformaldehyde at 4 o C overnight.Perilymph was collected prior to fixation using a 20 μl pipette placed in the round window after perforation of the round window membrane.Perilymph was placed in 1.5 ml microcentrifuge tubes, snap frozen in liquid nitrogen and stored at -80 o C for BDNF and NT3 ELISA.Samples from brain, heart, blood, mastoid process muscle, liver, kidney, stool and urine were collected and snap frozen in liquid nitrogen for subsequent PCR analysis.After fixation, cochleae were decalcified at 4 o C in phosphate buffered 8% EDTA (pH7.6) for 2 weeks.Cochleae from experiments using pFAR4-eGFP were hemi sectioned with a scalpel blade and whole-mount imaged on a LSM710 platform (Zeiss) to quantify the number of GFP transfected cells.Cochleae from deafened guinea pig experiments using pFAR4-CMVp-BDNF-NT3 to assess capacity for auditory neuron regeneration were cryoprotected in 30% sucrose prior to cryosection into 50 μm sections, and immunolabelling free-floating for TUBB3 (β3Tubulin; clone TUJ1 Covance) as previously described (Pinyon 2014, Pinyon 2016).Immunolabelled sections were mounted on coverslips using Prolong Gold mounting media (P36931; ThermoFisher) and imaged on a LSM710 platform with a 488 nm argon ion laser.Images were analyzed for neurite regeneration via fluorescence intensity of the TUBB3-labelled auditory neuron peripheral processes using ImageJ (US NIH).The segmented line tool was used to delineate the distal 50% of the osseus spiral lamina region (corresponding to the most pronounced region of neurite density loss at one month -post deafening.The average pixel intensity in this region was determined for sections from each of the four left (deafened with pFAR4-CMVp-BDNF-NT3 delivery) and the control right (deafened only) cochleae.

Cat tissue collection and analysis
Following electrophysiological recordings in the IC, animals were euthanized via intraperitoneal injection of 100 mg/kg pentobarbital and perfused systemically with heparinized 0.9% saline prior to 4% paraformaldehyde at 4 o C. Cochleae were isolated and fixed for an additional hour submerged in 4% paraformaldehyde at 4 o C. Cochleae were decalcified in 10% EDTA for 2 weeks then cryoprotected with 30% sucrose before freezing and cryosectioning at 50 μm.Free-floating sections were immunolabelled for TUBB3 (β3Tubulin; clone TUJ1 Covance) and NF200 (neurofilament protein 200 kDa; Cat# N4142, Merck) with respective secondary antibodies goat anti-mouse Alexa Fluor TM 488 (A-11001; ThermoFisher) and goat anti-rabbit Alexa Fluor TM 594 (A-11072; ThermoFisher).All antibodies were used at 1:1000 dilution.Sections were immunolabelled, mounted, imaged and analyzed for neurite regeneration as described above for the guinea pigs, with all sections containing the basal turn osseous spiral lamina (OSL) analyzed (n = between 7 and 10 sections per cochlea, with OSL areas measured between 32,334 and 57,259 square microns.Additionally, average fluorescence intensity of the auditory neuron somata was analyzed in ImageJ (US NIH) using the elliptical selections tool to outline each soma.Four sections from each cochlea were analyzed with between 9 and 61 somata measured from each section.Fluorescence intensity of both cell somata and neurites was measured in the 488 nm argon ion laser channel (TUBB3 immunodetection).At the time of euthanasia additional samples (blood, brain, heart, liver, kidney, lung, spleen, skin, intestine, and muscle from beside the ear canal) were collected and snap frozen in liquid nitrogen for PCR analysis.Samples then underwent DNA extraction and PCR analysis.

Montecarlo analysis of CC-GET electric field thresholds
A finite element model was developed to simulate the electric potential within the media surrounding the 8-electrode cochlea array in the in vitro HEK293 cell CC-GET model.Model input parameters include the media volume, the height of the media and the meniscus forming over the array (100 µm), the long semi-axis of the ellipse defining the meniscus, the supply voltage (120 V), and the voltage loss across the electrode-electrolyte bilayer, and the conductivity of the media.Experimentally indeterminate parameters were defined using a Monte-Carlo approach and were randomly selected from a uniform distribution to generate a population of models (n = 10), long-axis of the ellipse defining the meniscus (range 0.3-1.3mm), voltage loss across the bilayer were random (range 2 -30 V).This produced a media volume of 21-30 µl, approximating that used experimentally.For each model, the media specific conductivity was estimated by a parameter optimization routine that minimized the difference between the experimentally recorded total system resistance (4000 Ohm) and the model predicted total resistance.For each model (n = 10), the electric field threshold was then estimated by identifying a uniform perimeter symmetrically around the array that enclosed an area equal in size to the area of HEK293 cell transfection measured experimentally.

Measurement of recombinant BDNF and NT3 by ELISA
BDNF and NT3 levels in HEK293 cell culture supernatant (four days post CC-GET) and in guinea pig perilymph samples (2 weeks post CC-GET) were analyzed.ELISA-based quantification of BDNF (R&D Systems catalogue number DY248) and NT3 (R&D Systems catalogue number DY267) was performed as specified by the manufacturer, followed by colorimetric readout measuring absorbance at 415 nm in a FLUOstar microplate reader (PerkinElmer).BDNF and NT3 concentration was extrapolated against a serial dilution of the relevant purified neurotrophin standard.

PCR probe for tissue dispersion of neurotrophin miniplasmid
Samples were thawed and DNA was purified via the HotShot alkaline lysis method [6] .PCR was then performed on the extracted DNA to detect any possible undegraded plasmid DNA still present, using a forward primer located in the codon optimized BDNF coding sequence (5'-GGTTCATCAGGATCGACACTTCC-3') and reverse primer located in the IRES (5'-GGAATGCTCGTCAAGAAGACAGG-3').PCR was conducted using Taq Polymerase with Thermopol Buffer (New England Biolabs) at 97 o C for 2 min, followed by 40 cycles of 97 o C for 30 s, 64 o C for 30 s and 72 o C for 1 min with a final elongation of 72 o C for 5 min.

Supporting Information -Figures and Tables
Figure S1.Plasmid maps showing the design of neurotrophin, reporter, and optogenetic expression vectors.(A) pAd-CMVp-hrGFPnls, used as a reporter plasmid, containing the nuclear localized humanized renilla GFP (hrGFPnls) reporter coding sequence as the second cistron downstream of an internal ribosome entry sequence (IRES) and the rat brain derived neurotrophic factor (rBDNF) coding sequence driven by the cytomegalovirus promoter (CMVp).(B) pFAR4-CMVp-eGFP, a miniplasmid free of antibiotic resistance genes (pFAR) containing the enhanced GFP (eGFP) coding sequence.The pFAR4 plasmid vector enables production of plasmids lacking the conventional antibiotic resistance selection marker genes, pFAR4 plasmids are produced using a genetically modified E. coli strain that contains a nonsense mutation in the essential thymidylate synthase (thyA) gene generating its thymidine auxotrophy.pFAR4 plasmids encode a nonsense suppressor t-RNA that restores the ThyA open reading frame and bacterial growth, thus avoiding the use of antibiotic resistance marker for selecting plasmid-containing bacteria.These plasmids are compatible with clinical translation under EMA guidelines for gene electrotransfer, as they lack antibiotic resistance genes, thereby preventing inadvertent conferral of antibiotic resistance in commensal bacterial in the surgical field (after [7] ).Demonstrating the reduction in charge transfer achieved by CC-GET, only 5 mA was required to achieve 35 Va using the sucrose carrier, compared with 50 mA current pulses for the same Va when using saline carrier.Data reflected in US patent ref. [8] Right-side box plots report on a 24 hour cell culture study using the same GET parameters for sucrose vs. saline carriers.This replicated the finding of the 3 day cell culture study where sucrose carrier similarly achieved a larger area of transduced cells.This experiment included DAPI staining post-fixation to enable cell counts of regions of interest (ROI) (B,C; whole field (Olympus Slideview VS200) images showing ROI, detailed in adjacent confocal LSM images, for saline and sucrose carriers respectively).(D) These 24 hour cell culture data indicated an equivalent relative gene expression efficiency using saline and sucrose carriers, against a background of lower cell counts in the saline carrier condition (two way ANOVA with Holm-Sidak post-hoc comparison).(E,F,G) demonstrate the impact of the saline carrier for GET on cell viability using the same GET conditions, with propidium iodide (2 µg/ml) added to the culture medium 1 hour after GET to label cells with irreversible electroporation / injury, followed by PFA fixation and imaging.E and F show whole field and detail confocal LSM images of the region in the vicinity of the gene delivery array placement on the HEK293 cell monolayer in saline and sucrose carrier conditions.(G) PI fluorescence delineating cell injury margin surrounding a mechanical artefact associated with contact of the electrodes with the cell layer (as delineated by the sucrose carrier area measurements, where PI signal is minimal) (t-test).on ranks comparing 15 mA with lower current levels. 1 mA produced minimal expression (P < 0.001 vs. 5 mA; t-test).Images from US patent ref. [8]

Figure S4. Longitudinal study of CC-GET -directed expression of the pFAR4-CMVp-eGFP reporter plasmid to the cells lining the guinea pig cochlea basal region perilymphatic scalae. The cell counts of GFP+ve mesenchymal cells (green) is highest 4 days
after gene electrotransfer and under-represents the total due to clustering.Data from 4 days to 16 weeks after in vivo delivery to the basal scala tympani chamber demonstrated significant attenuation in expression.pFAR4-CMVp-eGFP miniplasmid delivered using the clinical CC-GET device (10 x 100 µs x 50 mA pulses).(A) reconstructed confocal image stack of the scalae of the basal region of the cochlea achieved by removal of the lateral wall.ST, scala tympani; SV scala vestibuli.Red is autofluorescence.(B) Box plots of the counts of GFP expressing cells at time points out to four months following CC-GET delivery of the naked GFP-encoding DNA (25% and 75% boundaries, thin line reflects the median; individual data overlay; ANOVA P value reflects time-dependent decline in expression attributable to turn-over of the targeted mesenchymal cells [3] ; n = 15).A,C,E).There was no significant difference between thresholds at different positions along the cochlear implant arrays attributable to a neurotrophin DNA CC-GET effect (P = 0.755), while a pronounced position-dependent effect was evident (P < 0.001; two-way ANOVA), where thresholds declined towards the apex (electrode e14) of the cochlear implant array.Control of gene expression with fixed charge transfer using electric field 'lensing', via change in separation between linear extended electrode elements was used to model GET electric field threshold.Montecarlo modelling established the electric field threshold for DNA electrotransfer, utilizing data from GFP reporter expression (threshold biomarker) in HEK293 cell monolayers.The effect of separation of the CC-GET 'electrolens' was evaluated using a modified GET array where the four ganged electrodes for one pole of the multi-electrode array were substituted for a single longitudinally extended Pt/Ir electrode of 2 mm length x 400 µm diameter, with variation of the separation with the other electrode surface, on a silicone backbone (shown in insets).CC-GET was performed using a GFP reporter plasmid including a nuclear localization signal to discretely resolve HEK293 cells expressing the transgene construct.To optimally model 'real world' intracochlear delivery we used a modified 'conductivity clamping' carrier solution comprising 9% sucrose and 0.09% saline (pH 7.4, 0.5 mM NaOH), with 2 µg/µl pAd-CMVp-hrGFPnls plasmid DNA (~ 12 µS.cm - conductivity).The size of the transfected field of HEK293 cells was assessed using a fixed suprathreshold pulse train (10 x 100 µs, 400 µs interpulse interval, 30 mA square wave pulses; requiring 120 V applied; ~ 30 µC charge transfer; ~ 4 kΩ local resistance).For this fixed charge transfer, the area of transfected GFP+ve cells was found to be scalable by a factor of 4.4, from 7.13 ± 1.34 mm 2 to 31.08 ± 3.24 mm 2 , when the anode -cathode separation was varied between 0.2 mm and 4 mm respectively (A-D, F).ANOVA indicated the significant effect on gene expression of increasing the separation between the electrodes (P = 0.001).The use of two elongated electrode surfaces was found to accurately model the cochlear implant-style multi-electrode array, where the 0.4 mm separation resulted in an average area of GFP expressing cells of 23.20 ± 3.50 mm 2 ; not significantly different from that achieved using the reference 8 electrode GET array with equivalent separation between the gangs of four anodes and four cathodes (18.85 ± 3.33 mm 2 , P = 0.500 ANOVA, E, F). (F) Boxplots of areas of transfected cells relative to electrode separation show 25% and 75% boundaries, with 95% limits as bars; lines indicate median, dashed lines indicate means, data overlaid (n = 4 per group).(G) Montecarlo modelling of electric fields across the configurations (see Supporting Information -Materials and Methods).The dashed perimeter, established by comparison with the perimeter of the field of transfected HEK293 cells reflects the electric field threshold for DNA electrotransfer (∆Vf/∆d = 120.8± 20.2 V/cm; mean ± SD.).(H) The predicted electric field threshold (120.8V/cm) was validated in silico by further simulations using the clinical CC-GET array.The long-axis of the fluid ellipse around the electrodes defining the meniscus and the voltage loss across the bilayer were set as the mean of the n = 10 random values generated in (G), 0.861 mm and 14.6 V respectively.Volume of solution was 26 µl.The media electrical conductivity was taken as the average of values estimated in (G), 0.437 S/m.Supply voltage was 120 V.The data summarizes the validation simulations that accurately predicted the surface area of the field of cells with GFP reporter expression (suprathreshold GET electric field strength) using the different 'lens' separations (spacing between the opposing conductive surfaces).Images (A-D) modified from Patent ref.
pronounced with stimulation of cochlear implant electrode positions e6 -e9, which closely corresponds to the peak of the CC-GET electric field.The boxplot indicates 25% -75% limits and the different symbols reflect the average for each cat (solid black line is the median, white dashed line is the mean).(I) Boxplots of average stimulus thresholds across all 32 IC electrodes for bipolar stimulation at each of the 7 cochlear implant electrodes, for three cats, with neurotrophin treatment in one cochlea and eGFP -encoding DNA treatment in the opposite cochlea.The data show that the cochlear neurotrophin gene augmentation treatment results in an overall reduction in excitability, which is associated with sharpening of spatial tuning.are from patent ref. [8]

Fig. S1 :
Fig. S1: Plasmid DNA maps Fig. S2: Increase in area of transfected HEK293 cells expressing GFP following CC-GET at equivalent applied voltage using sucrose DNA carrier solution (conductivity-clamping) compared with saline carrier; efficiency and cell injury prevention Fig. S3: Determination for threshold for reporter plasmid expression with clinical CC-GET DNA electrotransfer using low conductivity-clamping via application of 10% sucrose DNA carrier solution Fig. S4: Longitudinal study of CC-GET -directed expression of the pFAR4 CMVp-eGFP reporter plasmid to the cells lining the guinea pig cochlea basal region perilymphatic scalae Fig. S5: Cochlear perilymph recombinant neurotrophin levels in deafened guinea pigs two weeks after CC-GET delivery of clinical pFAR4-CMVp-BDNF-NT3 DNA Fig S6: Electrically evoked compound action potential (ECAP) thresholds after CC-GET delivery of neurotrophin or control (eGFP) DNA to the cochleae of deafened cats, supporting long-term hearing using bilateral cochlear implants Fig. S7: Sustained regeneration of the peripheral spiral ganglion neurites in the cochleae of deafened cats supported by chronic cochlear implant stimulation following CC-GET delivery of neurotrophin-encoding DNA Fig.S8: Sharpening of inferior colliculus spatial tuning curves with cochlear CC-GET delivery of BDNF -NT3 neurotrophin DNA followed by chronic cochlear implant stimulation in the deafened cat (C) pFAR4-CMVp-BDNF-NT3 contains the same miniplasmid backbone as (B) but with human BDNF and neurotrophin-3 (NT3) cistrons separated by an IRES.(D) pBs-CMVp-mCHERRYnls depicts the reporter plasmid used for the experiments shown in Fig. 4. (E) pJL-hSYNp-ReaChR-tdTomato contains a fusion protein of the red-shifter channelrhodopsin (ReaChR) and 2x tdTomato reporter under the control of the human synapsin promoter (hSYNp).(F) pAG-CMVp-GCaMP5g depicts the genetically encoded Ca 2+ reporter.(G) pMK-CAGp-eGFP contains the eGFP reporter downstream of the cytomegalovirus-Chicken beta-actin-rabbit beta-globin hybrid synthetic promoter (CAGp).

Figure S2 .
Figure S2.Increase in area of transfected HEK293 cells expressing GFP following CC-GET at equivalent applied voltage using sucrose DNA carrier solution (conductivityclamping) compared with saline carrier; efficiency and cell injury prevention.(A) 3 day cell culture study (left box plot set): 3 x 100 ms pulses at 35 V applied (Va) to the gene electrotransfer (GET) probe.pAd-CMVp-hrGFPnls plasmid (2 µg/µl) in 10% sucrose (black circles), or in normal saline (0.9% NaCl) with (black circles) or saline + Tris Buffer (50 mM, pH7.4; open circles; data for saline experiments were pooled; P > 0.05, t-test).The size of fields of the nuclear localized GFP+ve cells within HEK293 cell monolayers after 3 days in culture were determined by drawing boundaries around the perimeter of reconstructed montages of confocal images.Box plots show 25% and 75% boundary layers with 95% limits, lines show

Figure S7 .
Figure S7.Sustained regeneration of the peripheral spiral ganglion neurites in the cochleae of deafened cats supported by chronic cochlear implant stimulation following CC-GET delivery of neurotrophin-encoding DNA.CC-GET delivery of pFAR4-CMVp-BDNF-NT3 DNA (BDNF-NT3) followed by continuous use of a cochlear implant device sustained the regenerated auditory nerve fibers (135 -159 days post CC-GET).The controls are the opposite cochleae where CC-GET delivered the pFAR4-CMVp-eGFP -encoding DNA (eGFP).TUBB3 (β III tubulin) immunofluorescence demonstrates enhanced neurite labelling in the osseous spiral lamina (OSL) and in the somata of the spiral ganglion neurons (SGN).HP, habenula perforata; SL, spiral limbus; ST, scala tympani; SV, scala vestibuli.

Figure S8 .
Figure S8.Sharpening of inferior colliculus spatial tuning curves with cochlear CC-GET delivery of BDNF-NT3 neurotrophin DNA followed by chronic cochlear implant stimulation in the deafened cat.Data are recorded from the same 32 channel array inserted into the inferior colliculus, with stimulation across seven cochlear implant electrodes for (A) each of pFAR4 CMVp-BDNF-NT3 CC-GET -treated cochlea and (B) the contralateral (control) pFAR4 CMVp-eGFP CC-GET -treated cochlea.Following the bilateral CC-GET with (neurotrophin (Nt) encoding DNA into one cochlea and control (eGFP) DNA into the opposite cochlea, the cats received bilateral cochlear implants (CI), which were switched on two weeks after the surgery and remained active for the duration of the study (C516, 135 days; C518 136 days).Data from the third cat is shown in Figure 6.Stimulus threshold of the evoked field potentials was determined as bipolar CI stimulus level (CL, current level).

3 *Figure 1 .
Figure 1.Non-conductive sucrose carrier amplifies bionic array electric field strength.This figure demonstrates the effect of substituting a standard saline (0.9% NaCl) -based carrier for plasmid DNA GET, with isotonic 10% sucrose.(A) 8-electrode GET array depicting four ganged anodes (+) and cathodes (-), where this longitudinal extension of the electrode poles contributes to the electric field 'lensing'.Each of the full ring Pt/Ir electrodes is 350 µm in width, 400 µm diameter, with 350 µm spacing.(B) Using a currentcontrolled stimulator, the voltage applied to the electrodes approaches the maximum driving voltage of the stimulator with minimal current when sucrose is the carrier.Addition of DNA (sheared salmon sperm DNA, average size 2 kb, 2 µg/µl) to the normal saline carrier (0.9% NaCl) reduces conductivity, so that the voltage applied (Va) to the electrode increases, which benefits GET, however, there is no appreciable additional effect of the DNA within sucrose carrier.(C) Measured at a single point relative to the array (e7, 0.5 mm), the rate that the sampled field voltage (Vf) increases with increasing Va is enhanced for DNA + saline, compared with saline alone (augmenting the advantage for GET at the level Va shown in (B)); note however, that conductivity clamping with sucrose produces a further augmentation of the Vf, for a given Va.(D) Extending from the point measurement of Vf in (C), here Vf is measured along the length of the array, 0.5 mm lateral.The greatest change in Vf with distance (maximum electric field strength; ∆Vf/∆d) occurs across the null point (Vf = 0 V) at the transition between the anode and cathode poles.This highlights the point that the absolute voltage (in the field) is irrelevant, and this close-field electric 'lensing' effect provides compression of the electric field not achievable with conventional GET electrode configurations due to current spread.(E) The isopotential map (hemifield; upper graphic) represents extended sampling of Vf within the region around the electrode array with sucrose carrier, enabling derivation of the (2D) electric field map (at Va = 40 V, lower graphic), which is circular and equivalent to suprathreshold GET field strengths for several mm orthogonal to the array.

Figure 2 .
Figure 2. Electric field 'lensing' via the CC-GET array enables spatial focusing of gene expression.Control of gene expression with fixed charge transfer using electric field 'lensing', via change in separation between linear extended electrode elements was used to model GET electric field threshold.Montecarlo modelling established the electric field threshold for DNA electrotransfer, utilizing data from GFP reporter expression (threshold biomarker) in HEK293 cell monolayers.The effect of separation of the CC-GET 'electrolens' was evaluated using a modified GET array where the four ganged electrodes for one pole of the multi-electrode array were substituted for a single longitudinally extended Pt/Ir electrode of 2 mm length x 400 µm diameter, with variation of the separation with the other electrode surface, on a silicone backbone (shown in insets).CC-GET was performed using a GFP reporter plasmid including a nuclear localization signal to discretely resolve HEK293 cells expressing the transgene construct.To optimally model 'real world' intracochlear delivery we used a modified 'conductivity clamping' carrier solution comprising 9% sucrose and 0.09% saline (pH 7.4, 0.5 mM NaOH), with 2 µg/µl pAd-CMVp-hrGFPnls plasmid DNA (~ 12 µS.cm - conductivity).The size of the transfected field of HEK293 cells was assessed using a fixed suprathreshold pulse train (10 x 100 µs, 400 µs interpulse interval, 30 mA square wave pulses; requiring 120 V applied; ~ 30 µC charge transfer; ~ 4 kΩ local resistance).For this fixed charge transfer, the area of transfected GFP+ve cells was found to be scalable by a factor of 4.4, from 7.13 ± 1.34 mm 2 to 31.08 ± 3.24 mm 2 , when the anode -cathode separation was varied between 0.2 mm and 4 mm respectively (A-D, F).ANOVA indicated the significant effect on gene expression of increasing the separation between the electrodes (P = 0.001).The use of two elongated electrode surfaces was found to accurately model the cochlear implant-style multi-electrode array, where the 0.4 mm separation resulted in an average area of GFP expressing cells of 23.20 ± 3.50 mm 2 ; not significantly different from that achieved using the reference 8 electrode GET array with equivalent separation between the gangs of four anodes and four cathodes (18.85 ± 3.33 mm 2 , P = 0.500 ANOVA, E, F). (F) Boxplots of areas of transfected cells relative to electrode separation show 25% and 75% boundaries, with 95% limits as bars; lines indicate median, dashed lines indicate means, data overlaid (n = 4 per group).(G) Montecarlo modelling of electric fields across the configurations (see Supporting Information -Materials and Methods).The dashed perimeter, established by comparison with the perimeter of the field of transfected HEK293 cells reflects the electric field threshold for DNA electrotransfer (∆Vf/∆d = 120.8± 20.2 V/cm; mean ± SD.).(H) The predicted electric field threshold (120.8V/cm) was validated in silico by further simulations using the clinical CC-GET array.The long-axis of the fluid ellipse around the electrodes defining the meniscus and the voltage loss across the bilayer were set as the mean of the n = 10 random values generated in (G), 0.861 mm and 14.6 V respectively.Volume of solution was 26 µl.The media electrical conductivity was taken as the average of values estimated in (G), 0.437 S/m.Supply voltage was 120 V.The data summarizes the validation simulations that accurately predicted the surface area of the field of cells with GFP reporter expression (suprathreshold GET electric field strength) using the different 'lens' separations (spacing between the opposing conductive surfaces).Images (A-D) modified from Patent ref.

Figure 4 .
Figure 4. Validation of recombinant BDNF and NT3 protein synthesis in HEK293 cells using the clinical CC-GET device to deliver pFAR4-CMVp-BDNF-NT3 miniplasmid and pBs-CMVp-mCHERRYnls reporter DNA.The plasmid DNAs were delivered as a 30 µl cocktail (1 µg/µl each) in 9% sucrose + 0.09% NaCl + 0.5 mM NaOH carrier, and CC-GET performed using an 8 node electrotransfer array in tandem configuration.(A and B) Either 10 x 100 µs x 20 mA (400 µs interpulse interval) positive pulses, or 5 positive then 5 negative pulses, were utilized for CC-GET.Switching of electrode polarity within the pulse train provided a significant increase in mCherry reporter fluorescence, as well as increases in BDNF, (C) and NT3 (D) synthesis.This improved expression achieved by changing polarity may be attributable to the bias of DNA electrotransfer to the cathode-facing cell surface saturating binding efficiency.Overall, the use of alternating monophasic pulses achieved significant improvement of both BDNF and NT3 production (P = 0.001; ANOVA).ELISA analysis methodology is described in Supporting Information -Materials and Methods.Data reflected in Figure 12 Patent ref..[9] Cells were imaged and supernatant collected at 4 days post CC-GET.

Fig. 7 .
Fig. 7. CC-GET gene delivery to guinea pig brain achieves targeted optogenetic neuromodulation.Enablement of naked plasmid DNA electrotransfer to the brain via close-field conductance-clamping using sucrose carrier.(A) GFP-positive neurons in the nucleus of the tractus solitarius (NTS) region of the brainstem identified by confocal fluorescence imaging in a 50 µm cryosection taken 3 days after in vivo CC-GET (pFAR4-CMVp-eGFP plasmid; 10 x 100 µs x 50 mA).(B) Detail of eGFP-positive globus pallidus (GP) neurons imaged 7 days post CC-GET (pMK-CAGp-eGFP; 3 x 100 ms x 10 mA pulses requiring ~35V applied voltage; 50 µm cryosection).(C) Detail of a transfected GP neuron from the same experimental animal providing the data shown in (B).(D) A field of dual-plasmid transduced neurons within GP (pAG-CMVp-GCaMP5g + pJL-hSYNp-ReaChR-tdTomato; 10 x 100 µs x 50 mA; tdTomatotagged ReaChR channel rhodopsin expression shown (300 µm vibratome section imaged via LSM as an image stack at 4 µm z resolution (95 µm total depth, 3D projection).(E) Detail of dual expression of ReaChR channel rhodopsin (red channel) and GCaMP5g Ca 2+ reporter (green channel) from the same animal as experimental data shown in (D).Boxed insets highlight neurons co-expressing ReaChR and GCaMP5g.(F) Detail of regions of interest delineating three neurons co-expressing ReaChR and GCaMP5g, where the ReaChR ion channels were photo-activated.(G) Intracellular Ca 2+ responses to 561 nm pulsed laser activation of the three neurons shown in (F) measured via the GCaMP5g green fluorescence emission (488 nm excitation).(H) Peak GCaMP5g Ca 2+ fluorescence responses for 12 neurons with photo-activation of the co-expressed ReaChR channel rhodopsin.Images from (B) and (C)are from patent ref.[8]